Development of an acute ovine model of polycystic ovaries to assess the effect of ovarian denervation

Abstract

Introduction: Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype.

Methods: Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary.

Results: Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle.

Discussion: Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival.

Keywords: follicle; gonadotrophin; polycystic ovary syndrome; sympathetic nerve; tyrosine hydroxylase.

Figure 1

Illustration of the protocols used in the development and manipulation of a polycystic ovary. (A) Validation of the model. (B) Using the model to test the effect of ovarian denervation.

Figure 2

Sympathetic nerve supply to the ovine ovary. (A) Photograph of an ovine ovary (white arrow) in situ highlighting the neurovascular pedicle (red arrow). (B) Transverse section through the ovarian pedicle stained with tyrosine hydroxylase (brown) showing several discrete sympathetic nerves within the pedicle. (C) A large sympathetic nerve (brown) in the neurovascular pedicle. (D-F) Smaller sympathetic nerves (brown) within the pedicle. Scale bar (B–D) = 50 µm, (E, F) = 20 µm.

Figure 3

Sympathetic nerve supply within the ovine ovary. (A) Confocal staining of the hilar region of the ovary stained for tyrosine hydroxylase (sympathetic nerves) in green and CD-31 (endothelial cells) in red. An arteriole is highlighted by the white arrow. (B) A discrete sympathetic nerve (brown) within the ovarian stroma. (C) Nerves (brown) seen around blood vessels and a small preantral follicle (arrows). (D) Small arterioles with endothelial staining (red) with clear sympathetic nerves (green) surrounding in transverse view. (E) The plexus of sympathetic nerves (brown) around an arteriole in longitudinal view. (F) The presence of sympathetic nerves throughout the ovarian cortex and around small preantral follicles. Scale bar = 50 µm.

Figure 4

Sympathetic nerve supply to the ovary after transplantation. (A) Larger nerves (arrow) stained for tyrosine hydroxylase (brown) in the hilar region of the ovary post transplantation. (B) Plexus of sympathetic nerves (brown) around blood vessels in the medulla (arrow). (C) Sympathetic nerves in the cortex close to primordial follicles (arrow). Scale bar = 50 µm.

Figure 5

Denervation of the ovary using needlepoint diathermy. (A) Ovarian stroma stained for tyrosine hydroxylase (brown) highlighting sympathetic nerves that was histoscored blindly as 3. (B) Contralateral ovary stained for tyrosine hydroxylase after diathermy for denervation showing no specific immunostaining, with a histoscore of 0. (C) Blinded tissue score for immunostaining for tyrosine hydroxylase in control (C) ovary and diathermy (D) ovary. (D) Tissue noradrenaline concentrations in control (C) ovary and diathermy (D) ovary. (E) Significant correlation between noradrenaline concentrations and tissue immunostaining score for noradrenaline. Scale bar = 50 µm.

Figure 6

The effect of denervation on ovarian structure and function. (A) Peak estradiol before ovulation in control (C, n=4) and after ovarian denervation (D; n=4). (B) progesterone dynamics across the luteal phase after ovulation in control (C) and ovarian denervation (D) sheep. (C) Total progesterone secretion across the luteal phase in control (C, n=4) and after ovarian denervation (D; n=4). (D) Significant reduction in tissue immunostaining score and (E) ovarian noradrenaline concentrations after denervation (each ovary is analysed separately). (F) No significant difference in number of antral follicles or (G) preantral follicles in representative mid ovarian tissue section. (H) Representative immunostaining for cleaved caspase-3 (brown) identifing follicular atresia. (I) Quantification of antral follicles positive for cleaved caspase-3 in control ovaries (C) and after ovarian denervation (D). (J) Representative immunostaining for Ki67 (brown) identifying growing follicles. (K) Quantification of antral follicles positive for Ki67 in control ovaries (C) and after ovarian denervation (D). (L) Transcript abundance in ovarian stroma for key ovarian growth factors in control ovaries (C) and after ovarian denervation (D). ** P<0.01, *** P<0.005, n.s, not significant, scale bar = 50 µm.

Figure 7

Acute modelling of PCO. (A) Static image of optimal tomography whole ovarian scan with (B) the same area of the ovary after tissue sectioning and haematoxylin and eosin staining after FSH infusion with low LH. (C) Static image of optimal tomography whole ovarian scan with (D) the same area of the ovary after tissue sectioning and haematoxylin and eosin staining after FSH infusion with high pulsatile LH. (E) Cumulative follicles in the whole ovary based of size after FSH infusion with low LH (n=3). (F) Cumulative follicles in the whole ovary based of size after FSH infusion with high pulsatile LH (n=3).

Figure 8

The effect of ovarian denervation on acute model of PCO. (A) Average number of antral follicles per ovary after FSH infusion with high pulsatile LH (n=3), (B) or after FSH infusion with low LH (n=3), after sham procedure. (C) Average number of antral follicles per ovary after FSH infusion with high pulsatile LH (n=3), (D) or after FSH infusion with low LH (n=3), after bilateral ovarian denervation procedure.